Project Summary/Abstract Mitochondria are essential organelles that are most well-known for being cellular powerhouses, due to their role in oxidative phosphorylation (OXPHOS) and other metabolic pathways. In addition, they have diverse roles in other areas of cell biology, including calcium handling, immunity, cell signaling, and formation of iron-sulfur clusters. Healthy mitochondria are therefore critical for human health, and many common diseases are associated with mitochondrial dysfunction. The broad goal of this application is to understand the mechanisms that control mitochondrial health. There are three mechanisms of particular interest. First, mitochondrial function depends on continual cycles of fusion and fission. These dynamic processes serve to homogenize the mitochondrial population within a cell and are critical for maintenance of the mitochondrial genome, morphology, and respiratory chain activity. Second, mitophagy is a major mechanism to recognize and remove dysfunctional mitochondria. Third, protein surveillance mechanisms exist to maintain the quality of the OXPHOS protein complexes that generate cellular energy. The OXPHOS complexes are composed of protein subunits encoded by two genomes--the nuclear genome and the mitochondrial genome--and therefore have unique challenges in achieving proper subunit stoichiometry and assembly. This research program targets gaps in knowledge in each of these three homeostatic mechanisms. For mitochondrial dynamics, this research program investigates the molecular mechanisms and physiological functions of fusion and fission. To understand molecular mechanism, structural studies are used to obtain atomic structures of the key molecules mediating these processes. An example is Opa1, the molecule that mediates inner membrane fusion. Little is known about how this molecule is able to bring two inner membranes together and mediate membrane merger. To understand physiological function, mouse studies will be used to determine the role of mitochondrial fusion and fission. The application highlights two biological systems--the astrocytes of the nervous system and the male germ cell--in which mitochondrial fission and/or mitophagy play a prominent role. In the case of male germ cell development, mutations in mitochondrial dynamics genes lead to distinct stage-specific defects, providing a biological system in which multiple pathways requiring mitochondrial dynamics can be deciphered. To understand how the quality of the OXPHOS complexes are maintained, innovative genetic screens in human cells will be used to identify pathways that sense and degrade excessive subunits. Such quality control mechanisms have been implicated in lifespan regulation. Taken together, these approaches will provide a deep understanding of homeostatic mechanisms that maintain mitochondrial health.